Unravelling the hidden power of esterases for biomanufacturing of short-chain esters

Microbial production of esters has recently garnered wide attention, but the current production metrics are low. Evidently, the ester precursors (organic acids and alcohols) can be accumulated at higher titers by microbes like Escherichia coli. Hence, we hypothesized that their ‘direct esterification’ using esterases will be efficient. We engineered esterases from various microorganisms into E. coli, along with overexpression of ethanol and lactate pathway genes. High cell density fermentation exhibited the strains possessing esterase-A (SSL76) and carbohydrate esterase (SSL74) as the potent candidates. Fed-batch fermentation at pH 7 resulted in 80 mg/L of ethyl acetate and 10 mg/L of ethyl lactate accumulation by SSL76. At pH 6, the total ester titer improved by 2.5-fold, with SSL76 producing 225 mg/L of ethyl acetate, and 18.2 mg/L of ethyl lactate, the highest reported titer in E. coli. To our knowledge, this is the first successful demonstration of short-chain ester production by engineering ‘esterases’ in E. coli.

. Schematic representation of the metabolic pathways for ester production and in silico Δ r G predicted for ester synthesis via esterification: (a) Ester bioproduction can be achieved via acyltransferase (using acyl-CoA and alcohol as precursors) or esterase (using organic acid and alcohol as precursors) catalysis. The metabolic pathway for engineering E. coli cells for the biosynthesis of short-chain esters (ethyl lactate and ethyl acetate) and precursors thereof, including ethanol, D-lactate, and L-lactate. The thickness of the arrows depicts the average flux in pathway 63 , dotted arrows indicate the esterification reaction through acyltransferase (AAT) or esterase (Est) routes. Heterologous enzymes (LDH, lactate dehydrogenase; ADH, alcohol dehydrogenase; PCT, propionyl CoA synthase; PDC, pyruvate decarboxylase) that have been overexpressed are represented in red color and bold, whereas the genes encoding for AAT, Est and lactate dehydrogenase have been enlisted in a green, orange, and blue box respectively next to the pathway. (b) Gibbs free energy (Δ r G and Δ r G′) estimation for varying reaction quotients (Q) at different temperatures (30 °C and 37 °C) for the production of ethyl acetate via an esterification reaction. Δ r G°, standard Gibbs free energy of a reaction; and Δ r G′°, standard Gibbs free energy of a reaction at pH 7 and ionic strength of 0.1 M. The image was created using Microsoft PowerPoint and Microsoft Excel. www.nature.com/scientificreports/ of a reaction at standard conditions) for the esterase catalyzed esterification; turned out to be negative, further adding to the conundrum (Fig. 1b). Therefore, experimental validation of the esterase catalyzed esterification route is important owing to the several advantages for rapid progress in ester synthesis. Comprehending this, under the current study, we have engineered E. coli by cloning various esterases, and have analyzed their in vivo esterification potential. The current work encompasses in silico mining of various esterases from Brettanomyces bruxellensis AWRI1499, Saccharomyces cerevisiae, Pichia pastoris, and Pseudomonas aeruginosa. The nucleotide sequences for the esterases identified from B. bruxellensis have incorrect non-nucleotide/non-amino acid inclusions in the online databases (NCBI Genbank and Uniprot) and hence, preliminary manual curation was performed based on their sequence homologies. Comparative biochemical analysis of all the enzymes unveiled that esterases could serve as potent esterifying biocatalysts. Furthermore, high cell density (HCD) fermentation and optimization of cultivation conditions showed significant production of ethyl lactate and ethyl acetate from the engineered cells at the laboratory as well as bioreactor scales. Therefore, our research is the first successful demonstration of using esterases to produce short-chain esters: ethyl lactate and ethyl acetate.

Results
Gene mining and sequence curation. Bioprospecting for esterifying enzymes was performed to obtain their genetic sequences from the following natural producers: B. bruxellensis AWRI1499; S. cerevisiae; Pichia pastoris; and Pseudomonas aeruginosa. As a first step, B. bruxellensis proteome was analyzed, and two esterases, acetylxylan-esterase (AXE2) and carbohydrate-esterase (CE) were identified for exploring their esterification potential 28,29 . Gene/polypeptide sequences for the selected enzymes from B. bruxellensis had incorrect nucleotide/amino acid inclusions in GenBank/ UniProt. Hence, manual curation was performed based on sequence similarities using blastn, blastp, and clustal omega tools (Supplementary Fig. SI-1a). Local alignment (BLAST) and multiple sequence alignment (ClustalO) of the GenBank/UniProt sequences were performed to obtain the most closely related sequences, and the incorrect inclusions were corrected by replacement with the nucleotide/ amino acids from the homologous sequence/s (Supplementary Fig. SI-1a). During replacement, the polarity and the hydrophobicity index of the neighboring amino acids were considered. Final nucleotide sequences were subjected to in silico translation (TRANSLATE tool) and analyzed for their folding using the SWISS-MODEL tool. Similarly, model fermentative yeasts Pichia pastoris and S. cerevisiae were explored for their inherent esterifying enzymes, acyl-coenzymeA: ethanol O-acyltransferase (AcAlT) and ethyl-ester-synthase-1 (EEB1), respectively.
Like yeasts, pathogenic bacteria like Pseudomonas are also known to synthesize potent esterases 30,31 . Interestingly, Esterase-A 32 is a membrane bound esterase from Pseudomonas with its inherent signal peptide, and it is completely annotated in protein repositories, including UniProt. Hence, EstA from Pseudomonas aeruginosa was also selected for studying its esterification potential 30 (Fig. 1a). Further, to increase its soluble cytoplasmic fraction, we truncated EstA by removing the signal sequence to produce tEstA 33,34 (Supplementary Fig. SI-1b). SDS-PAGE results of the soluble fraction showed a more prominent protein band corresponding to tEstA (~ 66 kDa) than EstA indicating successful solubilization ( Supplementary Fig. SI-1b). In total, four different esterases (EEB1, EstA, CE, and AXE2), along with an edited tEstA, and a representative acyltransferase (AcAlT) were considered for heterologous cloning into E. coli 35,36 (Fig. 1a).
In vitro estimation of esterase activity. All the genes coding for esterases and an acyltransferase were codon optimized, synthesized, and successfully cloned under the control of T 7 promoter (P T7 ). Phase I E. coli strains were constructed by transforming E. coli BL21-DE3 with the above plasmid constructs (Supplementary Fig. SI-2). The whole cell lysates of the induced Phase I strains were analyzed for ester hydrolysis activity based on pNPA colorimetric assay. k cat /K M (catalytic efficiency) and K M values revealed that EEB1 exhibited the highest catalytic efficiency for pNPA hydrolysis over other candidates (Fig. 2). AcAlT, despite being acyltransferase, showed the second highest efficiency and relatively lower K M (higher affinity for the substrate), presumably due to its broader substrate specificity (Fig. 2). CE and tEstA, on the other hand, indicated relatively moderate hydrolysis activity with higher K M values than that of AcAlT (Fig. 2). However, AXE2 exhibited lower catalytic efficiency, but higher K M comparable that of the BL21-pET lysate control. Therefore, all the enzymes were further investigated for their in vivo esterification potential.
Metabolic engineering of E. coli for in vivo esterification. As a next step to assess the esterification potential of these heterologous enzymes, the Phase I strains were investigated for the production of ethyl lactate. This part of the study leveraged on the inherent ability of E. coli to produce both ethanol and lactate. BL21-pET strain possessing empty plasmid was used as a negative control. Therefore, first, the control strain BL21-pET possessing empty plasmid (Table 1), was explored for the production of lactate and ethanol through high-cell density (OD 600 = 3.0, HCD3) fermentation. Results indicated that the strain could synthesize lactate (3.8 g/L) and ethanol (0.7 g/L) as expected ( Supplementary Fig. SI-3a). However, as the ethanol concentration was relatively low, preliminary screening of all the Phase I strains was performed with HCD3, with an external doping of 10 g/L ethanol. However, the ethyl lactate titers amongst the Phase I strains were very less (< 0.1 mg/L) indicating that sole external supplementation of the precursors may not be sufficient ( Supplementary Fig. SI-3b). Therefore, the Phase I strains were engineered with the pathways for overproduction of ethanol and lactate, forming Phase II strains ( Supplementary Fig. SI-2). The Phase II strains had overexpression of the genes for stereospecific lactate (gldA101 for D-lactate or lldD for L-lactate), and ethanol (pdc and adh genes from Zymomonas mobilis) production (Table 1).
Fermentation studies were conducted with the Phase II strains, to screen for ester production. The preliminary fermentation (OD 600 = 3.0, HCD3) results indicated that Phase II strains produced significantly higher ester titers  Fig. 3a). Based on the ester titers from the preliminary screening and considering the possible interference of atmospheric oxygen in fermentation during intermittent aliquot sampling, the Phase II strains were subjected to higher cell density fermentation (OD 600 = 10.0, HCD10) using a sample-wise cultivation strategy. Unlike aliquot sampling, where a small portion of the sample was removed for analysis from the same culture tube every time, sample-wise cultivation strategy employed a separate set of tubes for every sample variant and the replicate. The strategy was implemented with (Supplementary Fig. SI-4b) and without external supplementation of ethanol, and corresponding D-or L-lactic acid. Strains possessing gldA101 gene (SSL64, SSL74, SSL76, SSL79) produced more ethyl lactate than their counterparts possessing lldD gene (SSL65, SSL75, SSL77, SSL80) (Fig. 3b). The highest ethyl lactate titers were exhibited with doping by SSL74 containing CE (8.2 ± 0.4 mg/L) and SSL76 containing EstA (9.5 ± 0.4 mg/L) (Fig. 3b). A quick study conducted by analyzing the spent medium showed that despite increase in the lactate production by the Phase II strains (0.35 g/L in WT to 1.45 g/L in SSL74), there was no effect on the acetate (1.5 g/L in WT and 1.6 g/L in SSL74) produced from acetyl CoA 37 . This observation supported the fact that pyruvate to acetyl CoA flux was unaffected by the overexpression of lactate genes 38,39 . However, well-controlled reactor-scale studies were necessary to comprehend the maximum ester biosynthesis potential of these strains. Therefore, batch fermentation with 60 g/L of glucose in hybrid M9 media was performed in a bioreactor to evaluate the ability of SSL74 and SSL76 to produce the short-chain esters (ethyl lactate and ethyl acetate) under anaerobic conditions.
Bioreactor study with high cell density fermentation in 'batch' mode. Although all the earlier experiments were performed by inducing the seed or pre-culture before subjecting it to the high cell density fermentation, to the best of our knowledge, there was no literature available on the effect of pre-culture induction on fermentation titers. Therefore, we studied this effect at the bioreactor scale with SSL76 strain. The seed culture was grown aerobically in a 1 L bioreactor, induced with 0.1 mM of IPTG and incubated for 18 h at 30 °C. The induced pre-culture was subjected to high cell density (with initial OD 600 ~ 4 to 5) in a batch cultivation mode and was compared for ethyl lactate production with un-induced pre-culture fermentation. Batch fermentation with un-induced pre-cultures showed higher titer for short-chain esters in comparison to that with the induced pre-cultures, at all the time points (Fig. 4a). Hence, for further bioreactor fermentations, IPTG induction was used only during the production phase. Separate set of optimization studies was carried out and the optimal IPTG concentration of 0.1 mM was selected for induction under this study (Supplementary Fig. SI-6a). Detailed optimization studies are mentioned in the Supplementary information SI-6.
Uninduced pre-cultures of SSL74 and SSL76 were subjected to high cell density batch fermentation. The kinetics of glucose, biomass, succinate, lactate, acetate, ethanol, and short-chain esters under batch cultivation mode are shown in Fig. 4b-d. Results showed that short-chain esters production peaked at 29 h (ethyl acetate (25.7 mg/L) and ethyl lactate (4.5 mg/L)) and then leveled off at later stage (Fig. 4b). Similar trend was observed for production of precursors indicating the complimentary correlation of ester production with precursors production. At 20 h of E. coli SSL74 cultivation, 12.6 g/L of lactic acid was produced under anaerobic conditions www.nature.com/scientificreports/ along with 14 g/L, 6.5 g/L and 5.2 g/L of ethanol, acetic acid and succinic acid, respectively. After 20 h, production of precursors leveled off (Fig. 4c). Similarly, both precursors and esters productions peaked for SSL76 at 20 h as well (Fig. 4d). Secondly, 95% and 85% glucose was consumed at 20 h while ethyl lactate and ethyl acetate were constantly produced by E. coli SSL74 and E. coli SSL76 strains, respectively (Fig. 4c,d).
Bioreactor study with high cell density fermentation in 'fed-batch' mode. To maximize the short-chain esters titers, anaerobic dual-pulse fed-batch cultivation was conducted at pH 7 by addition of 25 g/L of glucose at 6 h and 20 h. As seen in Fig. 5, dual-pulse fed-batch cultivation approach resulted in more than three-fold increase in ethyl acetate production (from 25.7 to 80 mg/L) and two-fold increase in ethyl lactate production (from 4.5 to 9 mg/L) in comparison to batch mode cultivation (Fig. 5a,b). In contrast to batch mode, we observed constant increase in ethyl acetate and ethyl lactate production beyond 20 h. Lactic acid, acetic acid, and ethanol productions were also observed to be gradually increasing suggesting that the availability of higher precursors concentration favored the esters production by engineered strains.
As discussed earlier, pH could be one of the limiting parameters during high cell density cultivation and could significantly affect ester biosynthesis 40,41 . Both the strains, SSL74 and SSL76, were equally efficient in ester production (Fig. 5a,b). Hence, only SSL76 was selected as a representative strain for the further study. To analyze the effect of pH, SSL76 was used as a representative platform strain and fed-batch cultivation was conducted at pH 6. At 72 h, despite relatively lower titers of ethanol, lactate and acetate in the medium than those with pH 7.0 (Fig. 6b), and maintaining the same cell density and glucose feeding, there was two-fold increase in the ethyl lactate production (18.2 mg/L) and three-fold increase in ethyl acetate production (225 mg/L) over pH 7 was    6a and 5b). This suggested that pH was a crucial factor for short-chain esters production using esterases.

Discussion
Esters are one of the major aroma components in wine and beer, produced due to the action of yeasts such as Saccharomyces cerevisiae and Brettanomyces bruxellensis 1,29,42,43 . B. bruxellensis is a non-conventional yeast used in the fermentation of craft and specialty beers and natural wine for its ability to produce secondary metabolites like esters during fermentation. It enhances the taste and aroma of the final product 44 . Like yeasts, pathogenic bacteria, e.g. Pseudomonas are also known to synthesize potent esterases involved with a rhamnolipid synthesis which exhibits an important role in their swarming motility, pathogenicity, outer membrane, and biofilm formation 30,31 . Hence, as a first step, mining and bioprospecting of various esterifying enzymes was performed. In total, five different esterases (EstA from P. aeruginosa along and its truncated variant tEstA, AXE2 and CE from B. bruxellensis, EEB1 from S. cereviciase), and an acyltransferase, AcAlt from Pichia pastoris were selected and considered for heterologous cloning into E. coli 35,36 (Fig. 1a). Phase I strains were constructed by transforming E. coli BL21-DE3 with the plasmid constructs possessing only the esterifying enzyme gene under P T7 (Supplementary Fig. SI-2). pNPA assay, performed using the whole cell lysates of their induced biomass, exhibited the following trend of their catalytic efficiencies for ester hydrolysis; AXE2 < CE < tEstA < AcAlT < EEB1. In principle, lower catalytic efficiency for ester hydrolysis, would mean higher esterification potential. So, purportedly AXE2 must be a better esterase. However, AXE2 had a weaker affinity towards the substrate as observed from its K M value. Therefore, we investigated all the enzymes for their in vivo ester production. As a next step, the Phase I strains were investigated for the production of ethyl lactate. First, the inherent ability of the control strain, BL21-pET to produce ethanol and lactate was analyzed. As BL21-pET produced relatively less ethanol ( Supplementary Fig. SI-3a), preliminary screening of all the Phase I strains was performed with an external ethanol supplementation. Such exogenous supplementation could provide better acid:alcohol ratio, thereby driving the equilibrium to ester production 41 . However, they showed very low ethyl lactate titers indicating that sole external supplementation of precursors may not be sufficient. Increasing the intracellular levels of the precursors will be essential for improving the ester synthesis ( Supplementary Fig. SI-3b). Hence, two distinct www.nature.com/scientificreports/ metabolic modules were engineered into E. coli: for precursor (alcohol and organic acid) overproduction, and for esterification (Fig. 1a), forming Phase II strains (Supplementary Fig. SI-2, Table 1). The comparative analysis of Phase II strains with intermittent aliquot sampling confirmed the in vivo esterification potential of esterases, giving significantly higher ester titers than the Phase I strains at HCD3 (Fig. 3a). Further analysis of Phase II strains was performed with sample-wise cultivation (HCD10) strategy, to avoid any oxygen interference faced during aliquot sampling. It was done with and without external doping of ethanol, to estimate whether higher precursor concentrations could drive more ester biosynthesis 41 . The results supported this hypothesis (Fig. 3b, Supplementary information SI-4b), indicating that increase in the precursor concentrations could improve the ester titers and it can be very well achieved at the controlled bioreactor scale. To reduce competition between cell growth and metabolite production, the fermentation was carried out in two stages; the first aerobic growth stage where the cells were grown till mid-exponential phase, and the second stage was maintained under anaerobic conditions to increase the production of esters. Results also exhibited SSL74 (possessing CE) and SSL76 (possessing EstA) as the potent candidates for short-chain ester biosynthesis (Fig. 3b). In addition, the esterases were more selective for D-lactate over L-lactate as one of the esterification substrates (Fig. 3b). Multiple parameters; IPTG concentration, phase for induction, pre-culture incubation temperature, and fermentation temperature were optimized (Supplementary information SI-6) for improving the ester production. Ultimately, to analyze the esterification potency of these best performing esterases (CE and EstA) at higher scale and under controlled environmental conditions, scale-up studies were carried out with a bench-top bioreactor in batch mode. Initially, the effect of pre-culture induction on ester production was analyzed in the bioreactor. To the best of our knowledge, there was no literature available on this effect. For this experiment, the results showed that the uninduced pre-cultures produced higher titers of ethyl lactate with high cell density fermentation than the induced ones (Fig. 4a). The lower titers with induced pre-cultures could be associated with the preliminary metabolic burden, which may have led to decreased productivity [45][46][47][48][49] . Secondly, it is reported that high expression of heterologous proteins results in an adverse and unpredictable outcome on the induced cell development. www.nature.com/scientificreports/ In contrast, too low protein expression levels result into reduced encounter frequency between the recombinant protein and the precursors [45][46][47][48]50 . Hence, the bioreactor studies were carried out under optimized conditions and cultures were induced only during the production phase (Supplementary information SI-6, Fig. 4a). Despite optimally controlled parameters in batch fermentation, ethyl lactate titers obtained with SSL74 and SSL76 were almost equivalent to the ones obtained in the shake-flasks (Fig. 4b). These titers could be attributed to the substrate limitation and/ or pH changes during the batch mode 51 . Therefore, for further improvement in the ester titers, anaerobic dual-pulse fed-batch cultivation was conducted at pH 7, which resulted in the two-threefold improvement in the ester titers. The results indicated that pH maintenance and glucose supplementation resulted in gradual increase in precursor concentrations, thereby improving the ester production from the engineered strains (Fig. 5a-d). However, pH could be one of the limiting parameters during high cell density cultivation and could significantly affect ester biosynthesis 40,41 . Difference in the pH can lead to variable ionic forms of the enzyme active site, ultimately affecting the enzyme activity 52 . In addition, only undissociated acids can participate in an esterification reaction, and the lower pH leads to higher fraction of such undissociated acid molecules in the system 41 . Hence, optimal esterification can be achieved at lower pH. On the other hand, for E. coli, it was reported that the pH values of less than 6.0 would negatively affect the cell growth 52,53 . Therefore, for in vivo esterification, it was necessary to select an optimal pH at which the enzyme activity would be relatively high and at the same time without negatively affecting the cell growth 54 . Hence, a separate set of in vitro study was performed for pH optimization. The results exhibited the highest ester production at pH 6.0 (Supplementary information SI-8). Therefore, to analyze the effect of pH 6.0 on in vivo esterification, SSL76 was used as a representative strain, and dual-pulse fed-batch cultivation was conducted at pH 6. Our results supported the earlier finding, that at pH 6, the ethyl acetate titer improved threefold, whereas the ethyl lactate titer increased by twofold over those observed under pH 7 (Fig. 6a). This suggested that controlled lower pH and dual-pulse of glucose during fed-batch cultivation were crucial factors for increasing the esterase catalyzed short-chain ester production from the engineered E. coli.

Conclusion
In this study, we evaluated various esterases for their esterification potential to produce short-chain esters, ethyl lactate and ethyl acetate. The fermentation parameters were optimized at the flask as well as the bioreactor scales for producing esters at higher titers. Overall, the results revealed that abundant availability of metabolic precursors ethanol, lactic acid, and acetic acid favored the short-chain esters production, while pH played a critical role in maximizing the enzyme activity for product formation. It was evident that EstA (from P. aeruginosa), and CE (from B. bruxellensis) showed comparable efficiencies in short-chain ester production. Therefore, the fed-batch fermentation by SSL76 strain (possessing EstA) overexpressing the genes for both ethanol and lactate synthesis, resulted in 225 mg/L of ethyl acetate and 18.2 mg/L of ethyl lactate at pH 6. This is the highest reported ethyl lactate titer from an engineered E. coli. Further improvement in esterification rate via esterases can be achieved through protein engineering and/or reactive extraction approaches using organic solvents. Conclusively, our study has successfully demonstrated the esterification potential of esterases, and we envision that in the future this strategy can be extended to other established and emerging model microbial hosts.   Fig. SI-2). Dual chemical transformation was performed for Phase II strains possessing both the plasmids; one possessing lactate dehydrogenase gene, esterifying enzyme gene and pct in pET15b plasmid while the other possessing ethanol biosynthesis pathway genes in pACYDuet plasmid ( Supplementary Fig. SI-2). All the expression strains have been enlisted in Table 1

In vitro esterase assay
All the esterases were simultaneously screened 59 using p-nitrophenyl acetate (pNPA) as a substrate for hydrolysis. Phase I strains were grown in LB medium with 100 µg/mL carbenicillin and induced using 1 mM IPTG during mid-exponential phase. The cells were harvested after 18 h of growth at 37 °C at 180 rpm. The pellets were stored at − 80 °C and lysed by in-house designed lysis strategy using SoluLyse 60 . Protein concentration of the cell lysate was measured using Bradford's method 60 and was used as a crude enzyme for the esterase assay. 5 mM pNPA was prepared in 1:1 ethanol-water mixture. The assay was performed using reaction concentrations of 70 µg/ mL of lysate protein and 0. www.nature.com/scientificreports/ a bioreactor with working volume of 500 mL containing hybrid M9 medium, inoculated with high cell density culture (initial OD 600 was ~ 4 to 5). The pH was maintained at appropriate value by auto-addition of 12 N NaOH and 1 N H 2 SO 4 . A dual-pulse anaerobic fed-batch fermentation experiment was also conducted in bench-top bioreactors with 500 mL as the initial working volume where an additional glucose of 25 g/L was manually fed to the bioreactor at 6 h and 20 h. The rest of the fermentation conditions were identical to the batch fermentation experiment, unless otherwise specified.
Substrate and metabolite analysis. Glucose, acetic acid, lactic acid, and succinic acid analyses were performed on an Agilent 1100 HPLC equipped with an Aminex 87H column, a Diode-Array Detection (DAD) and a refractive index detector (RID). Culture broths were collected at appropriate time intervals and centrifuged (Eppendorf Centrifuge 5415 D) at 12,000 rpm and 2 min. The supernatants were collected, appropriately diluted, filtered and injected onto an ion exchange HPLC column (an Aminex 87H column). The mobile phase was 5 mM H 2 SO 4 at a flow rate of 0.6 mL/min. The column temperature was at 70 °C and the RID detection (for glucose quantification) temperature was at 37 °C and DAD detection (for acids quantification) at 210 nm. Enzyme based D(−)/L( +) lactic acid detection kit (R-biopharm) was used to verify the optical purity of lactate from the engineered strains.
GC-MS analysis. Ester were quantified using GC-MS analysis. For the analyses, analytes in the culture supernatants and/or whole cultures were extracted using chloroform in a 5:4 (v/v) ratio for 10 min at RT, on a vortex machine, in 2 mL microcentrifuge tubes. The mixture was centrifuged at 10,000 rpm for 2 min and the 1 µL of organic layer was injected into the GC system (7890A) coupled with 5975C inert MSD system from Agilent Technologies. The injection was made in a splitless mode. For GC system, hydrogen was used as a carrier gas with a flow rate of 1 mL/min and analytes were separated on DB-5ms column (